Metastatic breast cancer is the leading cause of death among women between the ages of 15 and 54 and affects approximately 13% of women during their lifespan. These can be grossly categorized as ductal or lobular depending on their site of origin in normal breast tissue. Tumors usually begin as non-invasive cells at the site of tumor origin, spread to surrounding tissue in the breast and eventually become fully metastatic and migrate to the lymph nodes and other parts of the body.
There is increasing evidence that cell-cell adhesion is a potent suppressor of metastatic breast cancer progression (Berx and Van Roy, 2001). For example, in infiltrating lobular breast carcinomas E-cadherin is often lost and the resulting disruption of adherens junctions initiates a complete dissolution of cell-cell adhesion which allows single cells to break away from the primary tumor and invade the stroma in a single file pattern (Cleton Jansen et al., 2002). Alterations in cell adhesion are more subtle in infiltrating ductal carcinomas where invasion is characterized by the movement of clusters of cells into the stroma (Page and Simpson, 2000). In the latter situation adherens junctions are often present (Acs et al., 2001; Gillett et al., 2001) but there appears to be a general loss of polarity that is characterized by the mislocalization of apical markers such as MUC-1-(McGuckin et al., 1995; Mommers et al., 1999; Diaz et al., 2001; Rahn et al., 2001) that may be fueled by the disruption of tight junctions (Hoover et al., 1997; Kramer et al., 2000; Kominsky et al., 2003). While transcriptional repressors of E-cadherin expression have been identified (Cano et al., 2000; Guaita et al., 2002), little is known about the mechanism responsible for the disruption of tight junctions during breast tumor progression.
CD34 was initially identified over 20 years ago as an hematopoietic stem cell and vascular endothelial marker and has alternatively been proposed to act as an: 1) enhancer of proliferation, 2) a blocker of differentiation, 3) bone marrow homing receptor, 4) cell adhesion molecule, and 5) a blocker of cell adhesion (Fackler et al, 1996, Krause et al. Blood, 1996, Baumhueter et al. 1993). Deletion of the CD34 gene in mice has only served to fuel this debate as these mice are relatively normal with very subtle defects in hematopoietic and vascular function. The most clear-cut experiments suggest that CD34-type proteins can act as either pro-adhesive or anti-adhesive molecules depending on their glycosylation status (Satomaa, 2002, Baumhueter et al., 1993 and Bistrup et al., 1999).
Podocalyxin, (also called podocalyxin-like protein 1 (PCLP-1), Myb-Ets-transformed progenitor (MEP21) or thrombomucin) is a heavily sialylated and sulfated integral membrane glycoprotein that interacts with the actin cytoskeleton. It belongs to the CD34 family of sialomucins and is highly expressed on the surface of hematopoeitic progenitors, vascular endothelia and podocytes which form a tight junction-free epithelial meshwork that surrounds glomerular capillaries in the kidney (Kerjaschki et al., 1984; Kershaw et al., 1995; McNagny et al., 1997). Evidence suggests that the primary function of this molecule is to act as a type of molecular “Teflon ™” to block inappropriate cell adhesion. For example, as kidney podocytes begin to express podocalyxin they undergo a dramatic morphological shift from adherent, tight junction-associated monolayers surrounding the glomerular capillaries to a more modified cell layer lacking tight junctions and with extensive fully-interdigitated foot processes that are separated from each other by slit diaphragms. These podocalyxin-covered slit diaphragms form the primary filtration apparatus of the kidney. Deletion of the podocalyxin-encoding gene in mice results in the persistence of tight-junctions between podocytes, a lack of foot process formation and perinatal death due to anuria and high blood pressure (Doyonnas et al., 2001). Conversely, when podocalyxin is ectopically expressed in kidney epithelial cell monolayers, tight junctions and adherens junctions are both disrupted (Takeda et al., 2000). Thus, both gain-of-function and loss-of-function experiments suggest that podocalyxin acts as a tissue-specific anti-adhesin during normal kidney development (Takeda et al., 2001, Doyonnas et al., 2001).
Circumstantial evidence suggests that podocalyxin expression may be upregulated in a variety of neoplastic scenarios. For example podocalyxin was recently identified as the peanut agglutinin-binding tumor antigen gp200 expressed on human embryonal carcinomas. (Schopperle et al., 2002). In addition, the human podocalyxin gene (PODXL) has been assigned to chromosome 7q32-q33 (Kershaw et al., 1997), which places PODXL very close to the 7q35ter region that has been identified as a gain site by comparative genomic hybridization in ductal carcinoma in situ, infiltrating ductal carcinoma and in lymph node metastasis (Aubele et al., 2000). Thus, while it is not yet clear whether the PODXL gene is amplified in breast carcinoma, its expression may be unduly influenced by a nearby amplicon. Under anemic conditions the inventors have recently shown that Podocalyxin expression is upregulated in mouse erythroid progenitor cells (McNagny submitted unpublished obs). Therefore, podocalyxin expression may be similarly upregulated in necrotic breast carcinomas where hypoxia-regulated genes are transcriptionally activated (Adeyinka et al., 2002). If this is indeed the case, it would have functionally important implications as tumor hypoxia helps to drive solid tumor progression generally (Knowles and Harris, 2001) and ductal carcinoma progression specifically (Bos et al., 2003; Helczynska et al., 2003).
Using homologies present in the cytoplasmic tails of CD34 and podocalyxin, endoglycan was identified as a novel member of this family of glycoproteins. Endoglycan mRNA and protein were detected in both endothelial cells and CD34+ bone marrow cells (Sassetti et al., 2000). Endoglycan, like CD34 and podocalyxin can function as a L-selectin ligand. Endoglycan utilizes a different binding mechanism, interacting with L-selectin through sulfation on two tyrosine residues and O-linked sLex structures (Fieger et al., 2003).